The utility of DNA microarrays for characterizing genotoxicity.

Microarrays provide an unprecedented opportunity for comprehensive concurrent analysis of thousands of genes. The global analysis of the response of genes to a toxic insult (toxicogenomics), as opposed to the historical method of examining a few select genes, provides a more complete picture of toxicologically significant events. Here we examine the utility of microarrays for providing mechanistic insights into the response of cells to DNA damage. Our data indicate that the value of the technology is in its potential to provide mechanistic insight into the mode of action of a genotoxic compound. Array-based expression profiling may be useful for differentiating compounds that interact directly with DNA from those compounds that are genotoxic via a secondary mechanism. As such, genomic microarrays may serve as a valuable alternative methodology that helps discriminate between these two classes of compounds. Key words: biomarkers, gene expression profile, genetic toxicology, mechanism of action, toxicogenomics

Moving Forward in Human Cancer Risk Assessment

The goal of human risk assessment is to decide whether a given exposure level to a particular chemical or substance is acceptable to human health, and to provide risk management measures based on an evaluation and prediction of the effects of that exposure on human health. Within this framework, the current safety paradigm for assessing possible carcinogenic properties of drugs, cosmetics, industrial chemicals and environmental exposures relies mainly on in vitro genotoxicity testing followed by 2-year bioassays in mice and rats. This testing paradigm was developed 40 to 50 years ago with the initial premise that ¿mutagens are also carcinogens¿ and that the carcinogenic risk to humans can be extrapolated from the tumor incidence after lifetime exposure to maximally tolerated doses of chemicals in rodents. Genotoxicity testing is used as a surrogate for carcinogenicity testing and is required for initiation of clinical trials (Jacobs and Jacobson-Kram 2004) and for most industrial chemicals safety assessment. Although the carcinogenicity-testing paradigm has effectively protected patients and consumers from introduction of harmful carcinogens as drugs and other products, the testing paradigm is clearly not sustainable in the future. The causal link between genetic damage and carcinogenicity is well documented; however, the limitations of genotoxicity/carcinogenicity testing assays, the presence of additional non-genotoxic mechanisms, issues of species-specific effects, and the lack of mechanistic insights provide an enormous scientific challenge. The 2-year rodent carcinogenicity bioassays are associated with technical complexity, high costs, high animal burden as well as the uncertainty associated with extrapolating from rodents to humans. Additional frustrations exist because of the limited predictability of the 2-year bioassay and, in particular, with regard to the problem of the prediction of false positives. For instance, in the Carcinogenic Potency Project DataBase (CPDB) which includes results from chronic, long-term animal cancer tests with mice, rats, hamsters amounting to a total of 6540 individual experiments with 1547 chemicals, 751 of those chemicals or 51% have positive findings in rodent studies. Similarly, when one considers all chronically used human pharmaceuticals, some 50% induce tumors in rodents. Yet only 20 human pharmaceutical compounds have been identified as carcinogens in epidemiological studies, despite the fact that quite a large number of epidemiological studies have been carried out on these compounds, e.g. NSAID¿s, benzodiazepines, phenobarbital. This high incidence of tumors in bioassays has lead to questions concerning the human relevance of tumors induced in rodents (Knight et al. 2006; Ward 2008). In summary, dependency on the rodent model as a golden standard of cancer risk assessment is neglecting the high number of false positives and clearly has serious limitations. Consequently, there is a growing appeal for a paradigm change after "50 years of rats and mice". For instance, the current demands for volume of carcinogenic testing together with limitations of animal usage as initially stipulated by REACH (Combes et al. 2006) will require revolutionary change in the testing paradigm. For the purpose of developing a road map for this needed paradigm change in carcinogenicity testing, a workshop was held in August 2009 in Venice, Italy entitled ¿Genomics in Cancer Risk Assessment.¿ This workshop brought together toxicologists from academia and industry with governmental regulators and risk assessors from the US and the EU, for discussing the state-of-the-art in developing alternative testing strategies for genotoxicity and carcinogenicity, thereby focusing on the contribution from the ¿omics technologies. What follows is a highlight of the major conclusions and suggestions from this workshop as a path forward.JRC.DG.I.3-In-vitro method

Candidate biomarkers for the diagnosis and prognosis of drug-induced liver injury: An international collaborative effort

Current blood biomarkers are suboptimal in detecting drug-induced liver injury (DILI) and predicting its outcome. We sought to characterize the natural variabilty and performance characteristics of fourteen promising DILI biomarker candidates. Serum or plasma from multiple cohorts of healthy volunteers (n=192 and =81), subjects who safely took potentially hepatotoxic drugs without adverse effects (n=55 and =92) and DILI patients (n=98, =28, and =143) were assayed for microRNA-122 (miR-122), glutamate dehydrogenase (GLDH), total keratin 18 (K18), caspase cleaved K18 (ccK18), glutathione S-transferase alpha (GSTα), alpha fetoprotein (AFP), arginase-1 (ARG1), osteopontin (OPN), sorbitol dehydrogenase (SDH), fatty acid binding protein (FABP1), cadherin-5 (CDH5), macrophage colony stimulating factor receptor (MCSFR), paraoxonase 1 (PON1, normalized to prothrombin protein), and leucocyte cell-derived chemotaxin-2 (LECT2). Most candidate biomarkers were significantly altered in DILI cases compared to healthy volunteers. GLDH correlated more closely with gold standard alanine aminotransferase (ALT) than miR-122 and there was a surprisingly wide inter- and intra-individual variability of miR-122 levels among the healthy volunteers. Serum K18, OPN, and MCSFR levels were most strongly associated with liver-related death or transplant within 6 months of DILI-onset. Prediction of prognosis among DILI patients using Model for End-stage Liver Disease (MELD) was improved by incorporation of K18 and MCSFR levels. Conclusion: GLDH appears to be more useful than miR-122 in identifying DILI patients. K18, OPN and MCSFR are promising candidates for prediction of prognosis during an acute DILI event. Serial assessment of these biomarkers in large prospective studies will help further delineate their role in DILI diagnosis and management. This article is protected by copyright. All rights reserved

Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans.

To assess the potential of individual bile acids (IBA) and their profiles as mechanistic biomarkers of liver injury for humans in real world situations, we interrogated samples collected under minimum controlled conditions (ie subjects were not fasted). Total bile acids (TBA) have been considered to be biomarkers of liver injury for decades, and more recently, monitoring of IBA has been proposed for differentiation of variety of etiologies of liver injury. We established a LC-MS/MS methodology to analyze nine IBA, generated reference ranges, and examined effects of age, gender, and ethnicity for each IBA. Furthermore, we evaluated the ability of IBA and their profiles to detect hepatic injury in subjects with a broad range of liver impairments. To date, our study utilized the largest total cohort of samples (N = 645) that were divided into 2 groups, healthy or liver impaired, to evaluate IBA as biomarkers. The TBA serum levels in the Asian ethnic group trended higher when compared to other ethnic groups, and the serum concentrations of IBA, such as glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), chenodeoxycholic acid (CDCA), and taurochenoxycholic acid (TCDCA) were significantly increased. To our knowledge, this report is the first to describe ethnic differences in serum concentrations of IBAs. In patients with hepatic impairments, with the exception of deoxycholic acid (DCA), the concentrations of IBAs were significantly elevated when compared with healthy subjects. The conjugated bile acids displayed greater differences between healthy subjects and subjects with hepatic impairments than non-conjugated bile acids. Furthermore, the subjects with hepatic impairments exhibited distinct profiles (signatures) of IBAs that clustered subjects according the nature of their liver impairments. Although additional studies are needed, our data suggested that the analysis of IBA has the potential to become useful for differentiation of various forms of liver injury